Cells having pluripotency such as embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) obtained via introduction of undifferentiated cell-specific genes into somatic cells have been reported (U.S. Pat. No. 5,843,780 or WO 2007/069666). Hence, as a method for treating myogenic diseases, and particularly, a method for treating muscular dystrophy, a therapeutic method involving transplanting skeletal muscle progenitor cells resulting from induction of the differentiation of pluripotent stem cells has recently received attention. Similarly, development of a therapeutic agent using homogenous skeletal muscle is also under consideration.
Here, methods for inducing the differentiation of ES cells into skeletal muscle progenitor cells or skeletal muscle have been developed as follows: (1) a method that involves proliferating a single human ES cell by suspension culture, culturing the resulting cells by adhesion culture in a serum free culture solution, isolating CD73 positive cells, further culturing the cells, isolating NCAM positive cells, and proliferating the cells (Barberi T, et al. Nat Med. 13: 642-8, 2007); and (2) a method that involves treating human ES cells with 5-Azacytidine (demethylating agent), culturing the treated human ES cells by suspension culture to form embryoid bodies, and then further carrying out adhesion culture (Zheng JK, et al. Cell Res. 16:713-22, 2006), for example.
However, these methods are problematic in that: it is necessary to isolate cells several times and although engraftment of induced skeletal muscle can be confirmed by transplantation into model mice, the resulting amount of the skeletal muscle is extremely low.